Characterization of Water Permeability of Beta vulgaris Root Protoplast

KARINA ALLEVA, MOIRA SUTKA, MARIO PARISI AND GABRIELA AMODEO

Iguazú, Misiones, Argentina

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigaciones Bioquímicas; 2004

SAIB

Previous works showed that water transport studies performed in a purified fraction of root plasma membrane vesicles of Beta vulgaris are an excellent approach for characterising water channel properties. Nevertheless, in this model the cytoplasmic machinery of the cell is disrupted. In order to study water transport at a cellular level, swelling assays must be performed in protoplasts. To achieve this strategy, Beta vulgaris root protoplasts were isolated from the root parenchyma and exposed to a hypoosmotic shock employing two different methodologies: one in which protoplasts were held with a glass pipette during the osmotic shock (microchamber technique) and other where protoplasts were transferred from iso to hypoosmotic solution (step changes). In both cases, volume changes after osmotic shock were followed by light microscopy to calculate osmotic water permeability coefficient (Pf). Pf was 25 ± 4 ìm.s-1 (n=18) and 24 ± 8 ìm.s-1 (n=22) respectively, showing no differences between both methodologies. Permeability was not altered in the presence or absence of 5 mM K+ in the external solution, but was dramatically reduced by 10 mM external Ca+2. In accordance with our previous results in isolated membranes, calcium reduces water permeability also at a cellular level, proving to be an important regulatory mechanism of water transport in Beta vulgaris root cells